1. Field of the Invention
This invention relates to a polypeptide having thrombin inhibiting activity and to a process for the manufacture thereof.
2. Description of the Prior Art
Thrombosis has attracted considerable attention as a disease exhibiting a tendency to increase lately. A thrombus is a blood clot which is generated by coagulation in the blood stream and the clinical phenomena in which thrombi are formed is called thrombosis. It is known that the thrombi are frequently formed at sites where changes in the vascular endothelium, especially sclerotic or inflammatory changes, occur; furthermore, these lesions rapidly increase with aging, and consequently the worldwide increase in life span is one of the causes of the increase in the incidence of thrombosis. Thrombi are known to be formed due to the deposit of fibrin in the microvascular system as a result of the pathological activation of thrombin in the blood of the whole body.
Thrombosis leads to vascular constriction and obstruction due to thrombi, which result in ischemic lesions and infarction in major organs such as the heart, brain and lungs and cause functional disorders thereof. Furthermore, these kinds of thrombosis have lately attracted attention as developmental pathology of organic inflammations due to immunological mechanisms, such as nephritis and pneumonitis like and accessory lesions associated with transplantation of organs and vessel substitutes. Furthermore, disseminated intravascular coagulation (DIC) syndrome, known as a pathological state in which thrombi frequently occur mainly in the microvessels, attracts attention as a peculiar symptom. The concept of DIC was first suggested in 1960s; in those days, DIC was considered to be a very rare syndrome. However, it has lately been revealed that DIC is not peculiar; moreover, bleeding generated in the late stage of various disorders and various clinical symptoms which had been overlooked without being well understood as organic symptoms have been understood as a result of DIC.
Examples of clinical pathology of thromboses are cerebral apoplexy, cardiac infarction, deep phlebothrombosis, obstruction of an extremity artery, pulmonary thrombosis and fundus thrombosis. Both overall morbidity and mortality of these thromboses of various organs in individual special categories are said to be in the first rank among various diseases.
Therefore, the clinical and pathological significance of thrombosis is considered to magnify its importance daily.
Examples of known therapeutic agents for thrombosis are heparin which acts via antithrombin III and anti-vitamin K agents which inhibit biosynthesis of vitamin K dependent blood coagulation factors. Furthermore, another known thrombin inhibitor is a gabexate mesylate agent which is a non-peptide proteolytic inhibitor. Since this agent is also effective in inhibiting activity of enzymes such as plasmin, kallikrein and trypsin which have physiologically important significance, its use requires careful supervision.
Heparin, which has long been well known, is an anti-thrombin agent frequently used in the cases of thromboses represented by DIC; however, since its action is to accelerate anticoagulant activity of antithrombin III, heparin is considered to be ineffective for therapy in the cases where antithrombin III is decreased, for example in the cases of thromboses associated with DIC or nephrosis (see Reference 1). From these points of view, development of promising novel antithrombus agents for therapeutic uses is of great importance in therapeutic and prophylactic medicine.
Considerable hope is placed in HV1-type hirudin as an antithrombus agent having such pharmaceutical effects. The HV1-type hirudin is a polypeptide having thrombin inhibiting activity present in the salivary gland of medicinal leeches (Hirudo medicinalis). The HV1-type hirudin consists of 65 amino acid residues, and the presence of three intramolecular S--S bridges, which are essential for expression of thrombin inhibiting activity, is known as a configurational characteristic. In particular, the HV1-type hirudin has particularly high specificity in action against thrombin and prethrombin 2 (dissociation constant: 0.8.times.10.sup.-10) (see Reference 2), and only the activated factor IV other than thrombin is inhibited. Namely, the HV1-type hirudin does not inhibit enzymes other than those related to blood coagulation. Furthermore, the HV1-type hirudin is said to be extremely low in toxicity, nonantigenic and readily excreted from the kidney in the urine in a form having biological activity (see Reference 3).
Considering these aspects, HV1-type hirudin has the potential to be used as a very valuable prophylactic or therapeutic agent for thromboses including DIC in place of conventional antithrombosis agents.
Before the availability of recombinant DNA techniques, HV1-type hirudin was produced by direct extraction from leeches. However, in this method, a large number of starved leeches were required to obtain only a small amount of hirudin, and a considerably complicated purification steps and time were needed to obtain a crude hirudin preparation. For example, just to obtain a crude hirudin preparation with the purity as low as 10% also containing various contaminant proteins derived from the leeches, other than the HV1-type hirudin, processing included heat extraction of the homogenate of leeches starved for 2 to 3 weeks, ethanol precipitation, acetone fractionating precipitation, adsorption and desorption using bentonite and isoelectric precipitation were required. Furthermore, in order to obtain the HV1-type hirudin in a pure form using this crude hirudin preparation, ECTEOLA cellulose column chromatography, Sephadex CM-25 column chromatography and gel filtration using Sephadex G-25 had to be carried out, and the yield is reported to be less than 0.001% (see Reference 4). Since it is impossible to obtain the hirudin in quantities as described above, the therapeutic utilization of the hirudin which can be expected from excellent characteristics thereof has not been achieved at present.
Recently, it is possible to produce a large amount of heterologous gene products employing microorganisms as hosts using recombinant DNA techniques by expressing heterologous genes which are not naturally present in the microorganisms.
Processes of producing substances using recombinant DNA techniques using microorganisms as hosts can be generally divided into two categories: intracellular production and extracellular excretive production.
In the case of intracellular production, heterologous gene products can be effectively produced in the cells; however, there are problems such as degradation of the heterologous gene products by intracellular proteases, formation of inclusion bodies observed in the case of mass quantity production, and addition of methionine, which is a start codon for transcription, to the amino-terminal of the heterologous gene products. According to current research, it has been revealed that these problems can be solved by causing the heterologous gene products to be secreted outside the cells (see Reference 5). Furthermore, in the case of the extracellular production, the desired heterologous gene products can be purified easily, and marked reduction in possible contamination of foreign substances can be advantageously attained.
As has been mentioned above, it is important to produce heterologous gene products and have them excreted, in terms of producing desired substances.
Intracellular and extracellular productions have been reported also regarding HV1-type hirudin. In the intracellular production of HV1-type hirudin using Escherichia coli as a host, HV1-type hirudin having as little as 0.2 mg/l A660 of thrombin inhibiting activity was reported to be accumulated (see Reference 6). Thus, the HV1-type hirudin was accumulated only in a small amount probably because of the accumulation of inactive hirudin where the S--S bond(s) which is essential for the expression of the thrombin inhibiting activity is not precisely crosslinked.
Where the expression is carried out within the cells to produce a preprotein in which a secretion signal is bound upstream of the N-terminal of HV1-type hirudin and the product is secreted outside the cells, problems due to the intracellular production can be avoided so that the HV1-type hirudin is expected to be secreted outside the cells.
From these points of view, the secretory productions of HV1-type hirudin using E. coli or yeasts as hosts have been reported. In the secretory production of HV1-type hirudin using E. coli as a host, particularly, H. Dodt et al. encountered a problem (see Reference 7). They tried to construct a secretion plasmid having a DNA sequence coding for the secretion signal of E. coli alkaline phosphatase, immediately followed by a DNA sequence coding for the mature HV1-type hirudin and then attempted secretion of HV1-type hirudin using E. coli as a host. In this case, a polypeptide with an addition of three amino acids upstream of the N-terminal of the HV1-type hirudin, other than the HV1-type hirudin, was secreted. The thrombin inhibiting activity of this polypeptide having the additional amino acids is only to about 1/500 that of HV1-type hirudin.
Furthermore, in the secretory production of heterologous gene products using yeasts as hosts, in particular, the problem that amino acid residues at the C-terminal of the heterologous gene products are deleted, has been reported (see Reference 8). In fact, also in the secretory production of HV1-type hirudin using yeasts as hosts, 10 mg of HV1-type hirudin was accumulated in 1 liter of culture (see Reference 9). However, in this case, the presence of hirudin having reduced thrombin inhibiting activity, in which one or two amino acid residues at the C-terminal of the HV1-type hirudin were missing, was reported (see Reference 10).
In order to solve these problems, investigations have been made on the secretory production of HV1-type hirudin using bacterial strains of the genus Bacillus, which are capable of secreting a large quantity of proteins and are often used as industrial microorganisms for enzymes, amino acids, nucleic acids and the like, based on considerable prior experience. In particular, investigations have been made on methods using Bacillus subtilis as a host, among the bacteria of the genus Bacillus, on which a large number of genetic, biological, molecular biological, and applied microbiological knowledge has been accumulated. There have been several reports on attempts regarding extracellular secretion of heterologous gene products using Bacillus subtilis having such characteristics (see References 11 and 12); however, it has also been reported that secretory production of a large amount of proteins derived from eukaryotic organisms using Bacillus subtilis is not necessarily easy (see Reference 13).
In order to accomplish effective secretion of a desired heterologous gene product, it is important to select a combination of a selected mature protein and a secretion signal. In particular, since the ligation structure arises between the heterologous gene product and the secretion signal, namely the amino acid sequence of the junction region greatly effects on the secretory efficiency and the function of the secreted heterologous gene products, it is important to decide what kind of amino acid sequence is used at this junction region.
Palva et al. (see Reference 14) and Schien et al. (see Reference 15) have reported the effect of the alteration of an original amino acid sequence of the secretion signal due to the junction region between the secretion signal and a heterologous protein on the efficiency of the secretive production of the heterologous protein.
For example, Palva et al. attempted to bind a DNA fragment coding for mature interferon (IFN) immediately after the region encoding Ala-Val containing a cleavage site of the secretion signal of alpha-amylase, a secretory protein, of Bacillus amyloliquefacieno, via a junction region consisting of 5 amino acid residues (Asn-Gly-Thr-Glu-Ala) to secrete human IFN. In this case, the secretion signal was removed, but it has been reported that the secreted interferon was a fusion protein secreted and accumulated in a form in which one amino acid (Val) or 6 amino acids (Val-Asn-Gly-Thr-Gln-Ala) were added upstream of the N-terminal of mature interferon; the amount of these proteins was 0.5 to 1 mg per liter of culture. On the other hand, Schein et al. attempted to ligate a DNA fragment coding for mature interferon (IFN) immediately after the region coding for an amino acid of the secretion signal of the alpha-amylase as used by Palva et al. to secrete human IFN protein. However, it has been reported that, in this case, a large amount of an IFN precursor or mature IFN was retained within the cell membrane while the amount secreted into the medium was very little.
Furthermore, the relation between the structure of the cleavage site of a secretion signal and secretion efficiency as well as membrane transport have been discussed (see Reference 21). As a result, it has been determined that in correct cleavage of the cleavage site, the amino acid sequence of the cleavage site plays an important role.
The aforementioned facts suggest that the secretive productivity of a heterologous protein is possibly increased by reproducing an amino acid sequence of an original secretion signal in the junction region between the secretion signal and the heterologous gene product. A possible explanation is that the amino acid sequence of the cleavage site of the original secretion signal can be more easily cleaved by signal peptidase as compared to that of an altered cleavage site. In order to reproduce such a cleavage site of the original secretion signal, two kinds of methods were considered: a method in which the junction region having a nucleotide sequence necessary to reproducing an amino acid sequence of the original secretion signal was inserted between the regions coding for the C-terminal of the secretion signal and the N-terminal of the desired heterologous gene, respectively; and a method in which a DNA sequence coding for the N-terminal region of the heterologous gene was altered so as to reproduce the amino acid sequence of the cleavage site of the original secretion signal.
Also, in the secretion of the HV1-type hirudin, the aforementioned discussion related to the junction of the secretion signal, none of the results have been satisfactory.
In the two methods described above, in the case where an insertion region to reproduce the amino acid sequence of the cleavage site of the original secretion signal is constructed between the C-terminal of the secretion signal and the N-terminal of the HV1-type hirudin, a fusion protein containing extra amino acids at the N-terminal may be secreted (see Reference 14). In particular, it has been pointed out that, in the case of HV1-type hirudin, the presence of such additional amino acids induces a marked decrease in thrombin inhibiting activity as shown in the literature discussed above (see Reference 7).
Furthermore, also in the latter method, it is reported (see Reference 22) that the amino acid sequence of the N-terminal region of the HV1-type hirudin plays an important role in maintaining high thrombin inhibiting activity. It is also reported (see Reference 16) that the amino acid sequence of the N-terminal (residues 1-5) of HV1-type hirudin is related to the configurational maintenance of the C-terminal active site of HV1-type hirudin, the amino acid sequence of the N-terminal region of HV1-type hirudin plays an important role in exerting thrombin inhibiting activity, so that it is quite possible that this alteration causes a decrease in activity. It was difficult in the prior art technique to find out such alteration that high secretion efficiency can be attained without diverse effects on the activity. Furthermore, in this respect, effective alteration of the amino acid sequence has not yet been provided.